| The significance and purpose of our study is to screening the hydrogen-oxidizing bacteria that can promote the growth of plants and develop microbiological fertilizer to promote the sustainable development.And hydrogen-oxidizing bacteria were belonged to the PGPR,more and more attention had been paid to them.We used Medicago sativa rhizosphere in Shaanxi province of China to isolate and identify hydrogen-oxidizing bacteria.The objective was to perfect the culture apparatus,obtain the bacteria that had stronger ability to oxidize H2,deeply study their characteristic and identify them,screen the hydrogen-oxidizing bacteria that could promote the growth of wheat and quest for the growth promoting mechanism.The content and results were as follows:A gas-cycle incubation system(H2 treatment system) was used to provide stable H2, mixed with the air and control gas flow rate under 280 mL/min.The concentration of H2 was 4.16×10-4-2.42×10-3 mol/L.Hydrogen-oxidizing bacteria in the soil were enriched and then mineral salt agar medium(MSA) was used to separate and culture them.We finally identified 37 bacteria.Gas chromatography was used to assay the H2 oxidizing ability of these 37 bacteria.The result showed that 8 of them had the value of oxidize H2 over 2.44×10-4 mol/L,took up 25.93 %of all the strains.We initially identified them as hydrogen-oxidizing bacteria.The strain WMQ-7 and FMG-5 consumed the H2 over 12.28×10-4 mol/L,however,strain WMQ-7 had the strongest ability to oxidize H2,which was about 19.90×10-4 mol/L.According to the cell,colony form and biochemical characteristics,we identified the 8 strains as follows,Pseudomonas,Plesiomonas,Pimelobacter,Xanthobacter,Leminorella, Terrabacter,Rarobacte.We studied the optimal culture condition of strain WMQ-7,which showed that the optimal carbon source was glucose,the optimal temperature was 30℃and the optimal pH was 7.0.In the experiments of the 8 hydrogen-oxidizing bacteria effect on wheat,the culture plate showed that strain WMQ-7,FMG-3 and FMG-5 had strong ability to promote the growth of wheat.Wheat roots respectively increased 73.92%,74.78%and 58.26%.The length of sprout effect by the 3 strains all grew longer over 34.64%,however,the strain FMG-3 had the strongest effect and the length of sprout increased 67.64%.Dry biomass of wheat effect by strain WMQ-7,FMG-3 and FMG-5 respectively increased 95.24%,57.14%and 47.62% than the blank control.After the wheat ripe,the quantity of grain increased over 100%,which meant that strain FMG-5,WMQ-7 and FMG-3 could promote wheat harvest.Made the grain into flour and test the content of reducing sugar,total sugar and protein.Strain FMG-3 had the strongest ability to increase the sugar content.The content of reducing sugar and total sugar was respectively increased over 23.76%and 7.38%than the blank control.Strain FMG-5 had the strongest ability to increase the protein content,which increased 22.86%.We focused on the ACC deaminase and siderophore to study the mechanism why hydrogen-oxidizing bacteria can promote the growth of plants.Thin layer chromatography was used to find out the strain which could have ACC deaminase,and the result showed that strain WMQ-7 consumed the ACC and had ACC deaminase.We used ninhydrin reaction to test the ability of ACC deaminase;the result showed that enzyme activity of strain WMQ-7 was 0.671 U/μg.In the MSA liquid culture with glucose,ACC could be exhausted by strain WMQ-7 within 7 d.We used MSA-CAS and MKB-CAS testing culture plate to screen the strain which could produce siderophore.The result showed that only strain WMQ-7 had positive reaction.In MSA-CAS testing culture plate,the blue culture around WMQ-7 colony was changed into pink color,while that in the MKB-CAS testing culture plate was changed into yellow. UV-spectrophotometer was used to scan the sample between 220 nm-600 nm.We detected the siderophore produced by strain WMQ-7 was at 404 nm and the absorbance was 0.834.But, contrast to the standard sample 2,3-DHBA,they were different.So,we judged the siderophore produced by strain WMQ-7 was not the type of catecholamine.Ferroin assay was used to test the content of siderophore produced by strain WMQ-7,and indirect calculate it was 7.1996μg/mL.16S rDNA sequence of strain WMQ-7,FMG-3,FMG-5 was analyzed and built the phylogenic tree.The result showed these 3 hydrogen-oxidizing bacteria were much different from each other and the genetic distance was very far.Strain WMQ-7 was clustered together with Pseudomonas putida in phylogenetic tree,with the sequence identity of 99%.Its 16S rDNA was 1451 bp and GC content was 53.8%.From the physiology characteristic and inherited characteristic,strain WMQ-7 was finally identified as Pseudomonas putida (GenBank accession number EU807744).16S rDNA sequence of strain FMG-3 and FMG-5 was respectively 1034 bp and 1394 bp;GC content was respectively 51.7%and 54.45%. Stain FMG-5 was 94%similar to the rhizobium,while the 16S rDNA sequence of strain FMG-3 could not find a similar comparison in GenBank.This might indicate the strain FMG-3 was a new species,which would have great significance and value in further study.
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